Compositions and methods for diagnosing patients with acute atherosclerotic syndrome

ABSTRACT

The present invention is in the field of autoimmunity. More specifically, the present invention relates to the detection of autoantibodies to domain 4 of beta 2-glycoprotein I (β 2 -GPI) as an indicator for atherosclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. provisional patentapplication Ser. No. 60/833,757 filed Jul. 26, 2006 and U.S. provisionalpatent application Ser. No. 60/918,225 filed Mar. 14, 2007, each ofwhich is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention is in the field of autoimmunity. Morespecifically, the present invention relates to the detection ofautoantibodies to domain 4 of beta 2-glycoprotein I (β₂-GPI) as anindicator for atherosclerosis.

BACKGROUND OF THE INVENTION

IgA class autoantibodies to beta 2-glycoprotein I (β₂-GPI) have recentlybeen reported in patients with acute myocardial infarction and also inpatients with ischemic stroke (Ranzolin A, et al., Arg Bras Cardiol.83(2):141-4; 137-40 (2004); Kahles T, et al., Rheumatology 44(9):1161-5(2005); Staub H L, et al., Arg Neuropsiquiat 61(3B):757-63 (2003)). Astriking observation from two of these studies was that the IgA β₂-GPIautoantibodies were usually detected in patients that were negative forIgA anti-cardiolipin antibodies (ACA) (Ranzolin A, et al., Arg BrasCardiol. 83(2):141-4; 137-40 (2004); Staub H L, Arg Neuropsiquiat61(3B):757-63 (2003)). This finding is in sharp contrast to thatobserved in patients with anti-phospholipid syndrome (APS), where bothanti-β₂-GPI and anti-cardiolipin antibodies are usually positive.

β₂-GPI is a serum protein composed of five homologous domains numbered1-5 from the N terminus. Domains 1-4 are composed of ˜60 amino acidsthat contain a motif characterized by a framework of four conservedcysteine residues, which form two internal disulfide bridges (Lozier, J.et al., PNAS 81:3640-3644 (1984)). The fifth domain differs from domains1-4 in that it contains 82 amino acid residues with six cysteines. Thefifth domain contains the phospholipid binding site (Hunt, J., et al.,PNAS 90:2141-2145 (1993)).

Conflicting findings have been published concerning the domainspecificity of anti-β₂-GPI autoantibodies. For example, in one study, itwas shown that using recombinant-β₂-GPI and β₂-GPI domain-deletedmutants (Dms) expressed in insect cells, that anti-β₂-GPI autoantibodiesfound in serum samples from patients with APS recognize domain 1 ofβ₂-GPI (Iverson, G M, et al., PNAS 95:15542-15546 (1998).

There are also reports that IgG anti-β₂-GPI autoantibodies in patientswith APS recognize epitopes on domains 3, 4 and 5 of β₂-GPI (Blank, M,et al., PNAS 96:5164-8 (1999); Blank, M, et al., PNAS 96:5164-8 (1999);Koike T, et al., J. Autoimmun 15: 97-100 (2000); Yang C D, et al., APLARJ Rheumatol 1:96-100 (1997); Iverson, G M, et al., J. of Autoimmunity18:289-297 (2002); and McNeeley P A, et al., Thromb Haemost 86:590-5(2001)).

Accordingly, there is a need for an enhanced understanding of theantibody profiles exhibited by APS patients (cardiolipin IgGpositive/β₂-GPI IgG/IgA positive), and the differences between theseprofiles and the profiles exhibited by cardiovascular patients withacute ischemic disease (cardiolipin IgG/IgA negative/β₂-GPI IgApositive), which is based on differing domain-specificity of the APS andcardiovascular patient's antibodies. The present invention provides suchan enhanced understanding, and relates to the finding that IgAanti-β₂-GPI autoantibodies that bind to domain 4 are found in a highpercentage of patients with acute atherosclerotic syndrome (ASS).

SUMMARY OF THE INVENTION

The present invention relates, in part, to a method for diagnosing asubject suspected of having acute atherosclerotic syndrome (AAS)comprising the steps of: preparing an antigen comprising a polypeptidehaving an epitope from domain 4 of β₂-GPI; reacting the antigen with abiological sample from the subject; and detecting IgA antibodies in thesample that bind to the antigen.

The antigen according to the present invention may consist of domain 4in its entirety, or an antigenic fragment of domain 4, and/or it mayinclude all or a portion of the sequences of domains 2, 3 and 5.Accordingly, the phrase “an epitope from domain 4” intends that theepitope is recognized by an antibody that is selective for domain 4, inthat it binds preferentially to domain 4 when compared to the other 4domains. Thus, the epitope may be a polypeptide consisting of domain 4(or a fragment thereof) alone, or a combination of domains 2, 3 and 4;2, 3, 4 and 5; 3 and 4; 4 and 5; 3, 4 and 5; and/or fragments thereof.

As shown below, the domain 4 sequence consists of 56 amino acids. Giventhat the minimum number of contiguous amino acids from domain 4 is inthe neighborhood of 6 it is also possible to construct an antigen thatis a multimer (such as a dimer, trimer, etc.) of the domain 4 epitope,with the repeating units separated by non-interfering linking regionssuch as polyglycine and other small nonpolar amino acids. Such linkingregions may or may not include the naturally existing flanking sequencesof the epitope.

According to one embodiment, the invention is a method of diagnosing anacute atherosclerotic syndrome in a subject, comprising determining thepresence or absence of IgA domain 4-specific anti-beta 2-glycoprotein I(β₂-GPI) antibodies in said subject, wherein the presence of said IgAanti-β₂-GPI antibodies indicates that said subject has an acuteatherosclerotic syndrome.

In an alternative embodiment, the invention is a method of diagnosing anacute atherosclerotic syndrome in a subject, comprising the steps of: a.obtaining a sample from a subject suspected of having an acuteatherosclerotic syndrome; b. contacting the sample with a β₂-GPI antigencomprising a domain 4 epitope; and c. detecting the presence or absenceof IgA domain 4-specific anti-β₂-GPI antibodies that bind to the domain4 epitope; wherein the presence of said IgA domain 4-specificanti-β₂-GPI antibodies in said sample indicates that said subject has anacute atherosclerotic syndrome.

In yet another embodiment, the invention is a method of diagnosing anacute atherosclerotic syndrome in a subject, comprising the steps of: a.contacting a sample from a subject suspected of having an acuteatherosclerotic syndrome with an epitope derived from domain 4 of aβ₂-GPI antigen comprising the amino acid sequence of SEQ ID NO:5 withoutlinker sequences under conditions suitable to form a complex of theepitope and IgA domain 4-specific anti-β₂-GPI antibody; and b. detectingthe presence or absence of the IgA domain 4-specific anti-β₂-GPIantibody in the complex, wherein the presence of said domain 4-specificIgA anti-β₂-GPI antibodies in said subject indicates that said subjecthas an acute atherosclerotic syndrome.

The method for detecting the IgA anti-β₂-GPI antibodies can be by anyknown method, such as with a labeled anti-IgA antibody in a variety ofwell known assay formats such as an enzyme-linked immunosorbent assay.

In one embodiment, the method further includes determining the presenceor absence of anticardiolipin (aCL) antibodies in said subject, whereinthe presence of said domain 4-specific IgA anti-β₂-GPI antibodies insaid subject and the absence of anticardiolipin (aCL) antibodiesindicates that said subject has an acute atherosclerotic syndrome.

The domain 4 epitope may exist in the form of a variety of differentcombinations of domains and fragments thereof. For example, the domain 4epitope may consist of the known domain 4 fragment antigenic sequencethat is adjacent to domain 5 (Kasahara, et al.). It may also be in theform of the full domain 4 plus 5 sequences with all or portion of domain3, or it may be in the form of the full domain 3, 4 and 5 sequences withall or a portion of domain 2, or it may be in the form of adjacentfragments of domain 4 and 5, and so on. However, in a preferredembodiment, domain 1 is completely absent.

Other aspects of the invention are described throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts: Competitive inhibition of APS sample 6635 binding toβ₂-GPI by Recombinant β₂-GPI and Dms. A constant amount of antibody wasmixed with varying concentrations of inhibitor in wells coated withβ₂-GPI. Recombinant β₂-GPI and DMs were used as inhibitors. Upper panelmeasures inhibition of IgG antibodies. Lower panel measures inhibitionof IgA antibodies.

FIG. 2 depicts: Competitive inhibition of IgA anti-β₂-GPI of ACS-71 frombinding to β₂-GPI by recombinant β₂-GPI and deletion mutants. A constantamount of antibody was mixed with varying concentrations of inhibitorsin wells coated with β₂-GPI. Recombinant β₂-GPI and deletion mutantswere used as inhibitors.

FIG. 3 depicts: The entire sequence and tertiary structure of β₂-GPI.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in the field ofmolecular biology, immunology and medicine are extensively utilized. Inorder to provide a clearer and consistent understanding of thespecification and claims, including the scope to be given such terms,the following non-limiting definitions are provided.

When the terms “one,” “a,” or “an” are used in this disclosure, theymean “at least one” or “one or more,” unless otherwise indicated.

The term “antibody” refers to a molecule which is capable of binding anepitope or antigenic determinant. The term “antibody” includes wholeantibodies and antigen-binding fragments thereof, including single-chainantibodies. Such antibodies include human antigen binding antibody andantibody fragments, including, but not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. The antibodies may be from any animal origin includingbirds and mammals, e.g., human, murine, rabbit, goat, guinea pig, camel,horse and the like.

The term “antigen” refers to a molecule capable of being bound by anantibody or a T cell receptor (TCR) if presented by MHC molecules. Anantigen may be additionally capable of being recognized by the immunesystem and/or being capable of inducing a humoral immune response and/orcellular immune response leading to the activation of B- and/orT-lymphocytes. An antigen may have one or more epitopes (B- andT-epitopes). Antigens as used herein may also be mixtures of severalindividual antigens.

The term “antigenic determinant” refers to a portion of an antigen thatis specifically recognized by either B- or T-lymphocytes. Antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors. An antigenic determinant contains one or more epitopes.

The term “autoantigen” refers to a constituent of self that binds anautoantibody or that induces a cellular response.

The term “autoantibody” refers to an immunoglobulin, antigen specific Bcell surface receptor (surface immunoglobulin), or antigen specific Tcell receptor directed against self-protein, carbohydrate or nucleicacid.

The term “epitope” refers to a portion of an antigen that is recognizedby the immune system, specifically by an antibody (e.g., anautoantibody), B-cell, or T cell, and thus the particular domain, regionor molecular structure to which the antibody, B-cell or T-cell binds. Anantigen may consist of numerous epitopes while a hapten, typically, maypossess few epitopes.

The term “wild-type” refers to a gene or gene product isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene or gene product. In contrast,the term “modified” or “mutant” refers to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics, e.g., hypomethylation) when compared to thewild-type gene or gene product. It is noted that naturally occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics, such as physical and biological properties,when compared to the wild-type gene or gene product.

The term “native protein” refers to a protein that contains only thoseamino acids found in the protein as it occurs in nature. A nativeprotein may be produced by recombinant means or may be isolated from anaturally occurring source.

The term “fragment” means a peptide, polypeptide, or compound containingnaturally occurring amino acids, non-naturally occurring amino acids orchemically modified amino acids. The fragments may range in size fromtwo amino acid residues to the entire amino acid sequence minus oneamino acid.

The term “subject” refers to an animal, including, but limited to, anave, ovine, bovine, ruminant, lagomorph, porcine, equine, canine,feline, rodent or primate, for example a human. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa mammalian subject, particularly a human subject.

The term “sample” is used in its broadest sense. In one sense, it ismeant to include a specimen or culture obtained from any source, as wellas biological and environmental samples. Biological samples may beobtained from animals (including humans) and refers to a biologicalmaterial or compositions found therein, including, but not limited to,bone marrow, blood, serum, platelet, plasma, interstitial fluid, urine,cerebrospinal fluid, nucleic acid, DNA, tissue, and purified or filteredforms thereof.

The term “serum sample” refers to a biological sample comprising serum.It is understood that a serum sample for use in the present methods maycontain other components, in particular blood components. Thus, wholeblood samples, or blood samples which have been only partiallyfractionated or separated but which still contain serum, are considered“serum samples” for purposes of the present invention. One skilled inthe art can readily obtain serum samples, for example by usingconventional blood drawing techniques. Furthermore, the presence ofpreservative, anticoagulants or other chemicals in the serum sampleshould not interfere the detection of IgA β₂-GPI antibodies.

The term “control” or “control sample” refers to one or more sample,such as a serum sample, taken from at least one healthy blood donor. Itis understood that when the control comprises multiple samples, the IgAβ₂-GPI-specific antibody level can be expressed as the arithmetic mean,median, mode or other suitable statistical measure of the IgAβ₂-GPI-specific antibody level measured in each sample. Multiple controlsamples can also be pooled, and IgA β₂-GPI-specific antibody level ofthe pooled samples can be determined and compared to the subject'ssample.

Atherosclerosis (also referred to as arteriosclerosis, atheromatousvascular disease, arterial occlusive disease) as used herein, refers toa cardiovascular disease characterized by plaque accumulation on vesselwalls and vascular inflammation. The plaque consists of accumulatedintracellular and extracellular lipids, smooth muscle cells, connectivetissue, inflammatory cells, and glycosaminoglycans. Inflammation occursin combination with lipid accumulation in the vessel wall, and vascularinflammation is with the hallmark of atherosclerosis disease process.

The term “acute atherosclerotic syndrome” or “AAS” refers to severaltypes of cardiovascular problems including, but not limited to, acutemyocardial infarction, acute coronary syndrome, “carotid artery study,”and peripheral artery disease.

β₂-GPI is a serum protein composed of five homologous domains numbered1-5 from the N terminus. The primary and predicted tertiary sequence isshown in FIG. 3. Domains 1-4 are composed of ˜60 amino acids thatcontain a motif characterized by a framework of four conserved cysteineresidues, which form two internal disulfide bridges. The fifth domaindiffers from domains 1-4 in that it contains 82 amino acid residues withsix cysteines. The fifth domain contains the phospholipid binding site.

The amino acid sequence of domain 1 (SEQ ID NO.:2) as shown in FIG. 3,starting with the N-terminal end and ending with the small sequence thatlinks domain 1 to domain 2 (underlined) is as follows:

GRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLT GLWPINTLKCTPRV

The amino acid sequence of domain 2 (SEQ ID NO.:3) as shown in FIG. 3,starting with the N-terminal end of the small sequence that links domain1 to domain 2 (underlined) and ending with the small sequence that linksdomain 2 to domain 3 (underlined) is as follows:

TPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGK WSPELPVCAPII

The amino acid sequence of domain 3 (SEQ ID NO.:4) as shown in FIG. 3,starting with the N-terminal end of the small sequence that links domain2 to domain 3 (underlined) and ending with the small sequence that linksdomain 3 to domain 4 (underlined) is as follows:

APIICPPPSIPTFATLRVYKPSAGNNSLYRDTAVFECLPQHAMFGNDTIT CTTHGNWTKLPECREVK

The amino acid sequence of domain 4 (SEQ ID NO.:5) as shown in FIG. 3,starting with the N-terminal end of the small sequence that links domain4 to domain 3 (underlined) and ending with the small sequence that linksdomain 4 to domain 5 (underlined) is as follows:

REVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSLDGPEEIECTK LGNWSAMPSCKAS

The amino acid sequence of domain 5 (SEQ ID NO.:6) as shown in FIG. 3,starting with the N-terminal end of the small sequence that links domain4 to domain 5 (underlined) and ending with the C-terminal end is asfollows:

KASCKVPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTIEVPKCFKEHSSLAFWKTDASDVKPC

As used herein, it shall be understood that the term “domain X” alonerefers to a polypeptide with the amino acid sequence without theunderlined linker sequences identified above, but when terms such as“domain X/Y” or “domain X+Y” are used, it refers to a polypeptide withthe amino acid sequence of the two domains linked by the appropriateunderlined linker sequence identified above. Likewise, referring to the“domain X amino acid sequence of SEQ. ID NO. A without linker sequences”means the same as saying the polypeptide that includes all of the SEQ.ID NO. A amino acids, without the underlined linker sequences identifiedabove.

“Dms” or “domain-deleted mutants” nomenclature for domain deletionmutants uses numbers to indicate the presence of β₂-GPI domains, while adash symbolizes the domain is missing. Thus D--345 is the name given tothe recombinant protein that contains domains 3, 4 and 5 while lackingdomains 1 and 2.

The β₂-GPI proteins of the present invention may also be its variants.Unless otherwise indicated, the term “β₂-GPI” refers both to nativeβ₂-GPI proteins, as well as variants thereof. As used herein, β₂-GPIvariants are β₂-GPI proteins which comprises an amino acid sequencehaving one or more amino acid substitutions, deletions, and/or additions(such as internal additions and/or β₂-GPI fusion proteins) as comparedto the amino acid sequence of a native β₂-GPI proteins, but whichnonetheless retain β₂-GPI immunologically activity. Such functionally orimmunologically equivalent variants may occur as natural biologicalvariations (e.g., polypeptide allelic variants, polypeptide orthologs,and polypeptide splice variants), or they may be prepared using knownand standard techniques for example by chemical synthesis ormodification, mutagenesis, e.g., site-directed or random mutagenesis,etc. Thus, for example, an amino acid may be replaced by another whichpreserves the physicochemical character of the β₂-GPI proteins or itsepitope(s), e.g. in terms of charge density,hydrophilicity/hydrophobicity, size and configuration and hence preservethe immunological structure. “Addition” variants may include N- orC-terminal fusions as well as intrasequence insertion of single ormultiple amino acids. Deletions may be intrasequence or may betruncations from the N- or C-termini.

The variants may have from 1 to 3, to 5, to 10, to 15, to 20, to 25, to50, to 75, or to 100, or more than 100 amino acid substitutions,insertions, additions and/or deletions, wherein the substitutions may beconservative, or non-conservative, or a combination thereof.Additionally, the β₂-GPI proteins of the present invention may compriseat least 10, at least 12, at least 15, at least 20, at least 25, atleast 30, at least 35, at least 40, or at least 50 consecutive aminoacid residues of a native β₂-GPI protein. Such a variant is preferablyat least about 50%, at least about 60%, at least about 70%, at leastabout 80%, as lest about 90%, or at least about 95% identical to anative β₂-GPI proteins. Furthermore, the β₂-GPI variants may remainimmunologically active with an activity of over about 1%, over about10%, over about 25%, over about 50%, over about 60%, over about 70%,over about 80%, over about 90%, over about 95%, or over about 100% ofthe immunological activity of the native protein.

Conservative modifications to the amino acid sequence of a β₂-GPIprotein generally produce a polypeptide having functional and chemicalcharacteristics similar to those of the original β₂-GPI proteins. Incontrast, substantial modifications in the functional and/or chemicalcharacteristics of a β₂-GPI protein may be accomplished by selectingsubstitutions in the amino acid sequence of the β₂-GPI protein thatdiffer significantly in their effects on maintaining (a) the structure(secondary, tertiary, and/or quaternary) in the area of the substitutionor (b) the charge or hydrophobicity of the molecule at the target site.Amino acid sequence modifications can be accomplished by chemical andbiological peptide and protein synthetic methods that are well know inthe art.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are required. For example, amino acidsubstitutions can be used to identify important residues, to modulatethe biological activity of a β₂-GPI protein, e.g., the bindinginteractions with β₂-GPI-specific antibodies, or to decrease unwantednon-specific binding interactions with other molecules in a sample.Suitable amino acid substitutions include, but are not limited to,substituting Ala with Val, Leu, or Ile; substituting Arg with Lys, Gln,or Asn; substituting Asn with Gln; substituting Asp with Glu;substituting Cys with Ser or Ala; substituting Gln with Asn;substituting Glu with Asp; substituting His with Asn, Gln, Lys, or Arg;substituting Ile with Leu, Val, Met, Ala, Phe, or Norleucine;substituting Leu with Norleucine, Ile, Val, Met, Ala, or Phe;substituting Lys with Arg, 1,4-diamino-butyric acid, Gln, or Asn;substituting Met with Leu, Phe, or Ile; substituting Phe with Leu, Val,Ile, Ala, or Tyr; substituting Pro with Ala; substituting Ser with Thr,Ala, or Cys; substituting Thr with Ser; substituting Trp with Tyr orPhe; substituting Tyr with Trp, Phe, Thr, or Ser; and substituting Valwith Ile, Met, Leu, Phe, Ala, or Norleucine. The selection of an aminoacid for replacement can also be guided by its hydropathic index and/orhydrophilicity.

In addition, the polypeptide may be fused to a homologous polypeptide toform a homodimer or to a heterologous polypeptide to form a heterodimer.Heterologous polypeptides include, but are not limited to: an epitope toallow for the detection and/or isolation of a β₂-GPI fusion polypeptide,such as, polyhistine at either C- or N-terminal to ease thepurification; an enzyme or portion thereof which is catalyticallyactive; a polypeptide which promotes oligomerization, such as a leucinezipper domain; and a polypeptide which increases stability, such as animmunoglobulin constant region.

Fusions can be made either at the amino-terminus or at thecarboxyl-terminus of a β₂-GPI polypeptide. Fusions may be direct with nolinker or adapter molecule or may be through a linker or adaptermolecule. A linker or adapter molecule may be one or more amino acidresidues, typically from about 20 to about 50 amino acid residues. Alinker or adapter molecule may also be designed with a cleavage site fora protease to allow for the separation of the fused moieties. It will beappreciated that once constructed, the fusion polypeptides can furtherbe derivatized according to the methods described herein.

The β₂-GPI protein of the present invention may also be β₂-GPIderivatives, which is a chemically or biologically modified protein,including protein post-translation modification, such as acylation(i.e., acetylation or formylation), biotinylation, carboxylation,deamination, glutathionylation, glycosylation, lipidation (i.e.,farnesylation, gernylgeranylation, prenylation, myristoylation,palmitoylation, or stearoylation), methylation, phosphorylation,sulphation, fucosylation, and ubiquitination. Unless otherwiseindicated, the term “β₂-GPI protein” refers both to native proteins, andvariants and derivatives thereof. A protein derivative may be modifiedin a manner that is different in the type, number, or location of thepost-translation modification groups naturally attached to thepolypeptide. For example, a derivative may have the number and/or typeof glycosylation altered compared to the native protein. The resultingderivative may comprise a greater or a lesser number of N-linkedglycosylation sites than the native protein.

The β₂-GPI polypeptide may also be modified by the covalent attachmentof one or more polymers. Typically, the polymer selected iswater-soluble so that the protein to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. The polymer may be of any molecular weight and may bebranched or unbranched. The polymer each typically has an averagemolecular weight of between about 1 kDa to about 100 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, polyalkylene glycol (such as mono-(C₁-C₁₀)alkoxy-,aryloxy-polyethylene glycol, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, or polypropylene oxide/ethyleneoxide co-polymers), carbohydrate-based polymers (such as dextran orcellulose), polyoxyethylated polyols, and polyvinyl alcohols. Alsoencompassed by the present invention are bifunctional crosslinkingmolecules which can be used to prepare covalently attached β₂-GPIpolypeptide multimers.

In general, chemical derivatization may be performed under a suitablecondition by reacting a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of: (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby the β₂-GPIprotein becomes attached to one or more polymer molecules, and (b)obtaining the reaction products. The optimal reaction conditions mayvary depending upon the β₂-GPI protein selected and chemical reagentsused, and are generally determined experimentally. The PEGylation of apolypeptide may be carried out using any of the PEGylation reactionsknown in the art, including, but not limited to, acylation, alkylation,or Michael addition.

Diagnostic Assay

There are many different types of immunoassays suitable for use in thepresent invention. Any of the well known immunoassays may be adapted todetect the level of β₂-GPI-specific antibodies in a sample which reactwith the β₂-GPI antigens, such as, e.g., enzyme linked immunoabsorbentassay (ELISA), fluorescent immunosorbent assay (FIA), chemical linkedimmunosorbent assay (CLIA), radioimmuno assay (RIA), immunoblotting, geldiffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotope labels,for example), Western blots, precipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc. For a review of the differentimmunoassays which may be used, see: The Immunoassay Handbook, DavidWild, ed., Stockton Press, New York, 1994. A competitive immunoassaywith solid phase separation or an immunometric assay for antibodytesting is particularly suitable for use in the present invention. See,The Immunoassay Handbook, chapter 2.

In one exemplary embodiment of the invention, the diagnostic assay is animmunometric assay for detecting the level of β₂-GPI-specific antibodiesin a sample. In the immunometric assay, the β₂-GPI antigens areimmobilized on a solid support directly or indirectly through a captureagent, such as anti-β₂-GPI antibodies. An aliquot of a sample, such as aserum sample, from a subject is added to the solid support and allowedto incubate with the β₂-GPI antigens on the solid phase. A secondaryantibody that recognizes a constant region in the autoantibodies presentin the sample which have reacted with the β₂-GPI antigen is added. Whenthe subject is a human, this secondary antibody is an anti-humanimmunoglobulin. The secondary antibody which is specific for IgA, IgG,or IgM heavy chain constant regions, or combination thereof, may beemployed. After separating the solid support from the liquid phase, thesupport phase is examined for a detectable signal. The presence of thesignal on the solid support indicates that autoantibodies to the nativeβ₂-GPI proteins present in the sample have bound to the β₂-GPI antigenon the solid support. Increased optical density or radiolabeled signalwhen compared to the control samples from normal subjects correlateswith a diagnosis of APS in a subject.

Solid supports are known to those skilled in the art and include thewalls of wells of a reaction tray (e.g., microtiter plates), test tubes,polystyrene beads, magnetic beads, nitrocellulose strips, membranes,microparticles such as latex particles, glass or silicon chips, sheep(or other animal) red blood cells, duracytes and others. Suitablemethods for immobilizing nucleic acids on solid phases include ionic,hydrophobic, covalent interactions and the like. A solid support, asused herein, refers to any material which is insoluble, or can be madeinsoluble by a subsequent reaction. The solid support can be chosen forits intrinsic ability to attract and immobilize the capture reagent.Alternatively, the solid phase can retain an additional molecule whichhas the ability to attract and immobilize the capture reagent. Theadditional molecule can include a charged substance that is oppositelycharged with respect to the capture reagent itself or to a chargedsubstance conjugated to the capture reagent. As yet another alternative,the molecule can be any specific binding member which is immobilizedupon (attached to) the solid support and which has the ability toimmobilize the β₂-GPI antigen through a specific binding reaction. Themolecule enables the indirect binding of the β₂-GPI antigen to a solidsupport material before the performance of the assay or during theperformance of the assay.

The signal producing system is made up of one or more components, atleast one of which is a label, which generate a detectable signal thatrelates to the amount of bound and/or unbound label, i.e., the amount oflabel bound or unbound to the β₂-GPI antigen. The label is a moleculethat produces or which may be induced to produce a signal. Examples oflabels include fluorescers, enzymes, chemiluminescers, photosensitizersor suspendable particles. The signal is detected and may be measured bydetecting enzyme activity, luminescence or light absorbance. Radiolabelsmay also be used and levels of radioactivity detected and measured usinga scintillation counter.

Examples of enzymes which may be used to label the anti-humanimmunoglobulin include β-D-galactosidase, horseradish peroxidase,alkaline phosphatase, and glucose-6-phosphate dehydrogenase (“G6PDH”).Examples of fluorescers which may be used to label the anti-humanimmunoglobulin include fluorescein, isothiocyanate, rhodamines,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde,fluorescamine, and Alexa Fluor® dyes (that is, sulfonated courmarin,rhodamine, xanthene, and cyanine dyes). Chemiluminescers include e.g.,isoluminol. For example, the anti-human immunoglobulin may be enzymelabeled with either horseradish peroxidase or alkaline phosphatase.

Enzymes may be covalently linked to β₂-GPI antigen reactive antibodiesfor use in the methods of the present invention using well knownmethods. There are many well known conjugation methods. For example,alkaline phosphatase and horseradish peroxidase may be conjugated toantibodies using glutaraldehyde. Horseradish peroxidase may also beconjugated using the periodate method. Commercial kits for enzymeconjugating antibodies are widely available. Enzyme conjugatedanti-human and anti-mouse immunoglobulin specific antibodies areavailable from multiple commercial sources.

Biotin labeled antibodies may be used as an alternative to enzyme linkedantibodies. In such cases, bound antibody would be detected usingcommercially available streptavidin-horseradish peroxidase detectionsystems.

Enzyme labeled antibodies produce different signal sources, depending onthe substrate. Signal generation involves the addition of substrate tothe reaction mixture. Common peroxidase substrates include ABTS(2,2′-azinobis(ethylbenzothiazoline-6-sulfonate)), OPD(O-phenylenediamine) and TMB (3,3′,5,5′-tetramethylbenzidine). Thesesubstrates require the presence of hydrogen peroxide. p-Nitrophenylphosphate is a commonly used alkaline phosphatase substrate. During anincubation period, the enzyme gradually converts a proportion of thesubstrate to its end product. At the end of the incubation period, astopping reagent is added which stops enzyme activity. Signal strengthis determined by measuring optical density, usually viaspectrophotometer.

Alkaline phosphatase labeled antibodies may also be measured byfluorometry. Thus in the immunoassays of the present invention, thesubstrate 4-methylumbelliferyl phosphate (4-UMP) may be used. Alkalinephosphatase dephosphorylated 4-UMP to form 4-methylumbelliferone (4-MU),the fluorophore. Incident light is at 365 nm and emitted light is at 448nm.

The amount of color, fluorescence, luminescence, or radioactivitypresent in the reaction (depending on the signal producing system used)is proportionate to the amount of autoantibodies in a sample which reactwith the β₂-GPI antigens. Quantification of optical density may beperformed using spectrophotometric or fluorometric methods, includingflow cytometers. Quantification of radiolabel signal may be performedusing scintillation counting.

In another exemplary embodiment, the assay is a competitive immunoassay,which employs one or more β₂-GPI-specific antibodies that binds to thesame epitopes as the β₂-GPI-specific autoantibodies. In the assay, theβ₂-GPI-specific antibodies and the β₂-GPI-specific autoantibodies in asample compete for binding to the β₂-GPI antigens. Typically, a constantamount of a labeled antibody which is known to bind to β₂-GPI antigen isincubated with different concentrations of a sample from a subject. Theβ₂-GPI-specific antibodies may be monoclonal or polyclonal.

As described herein above, the β₂-GPI-specific antibodies may be labeledwith a fluorescer, enzyme, chemiluminescer, photosensitizer, suspendableparticles, or radioisotope. After incubation, bound labeled antibodiesare separated from free antibodies. Depending on the signal producingsystem used and if necessary, an appropriate substrate with which thelabeled antibody reacts is added and allowed to incubate. The signalgenerated by the sample is then measured. A decrease in optical densityor radioactivity from before and after addition of the serum sample orbetween experimental and control samples, is indicative thatautoantibodies in the sample have bound to the β₂-GPI antigen. Decreasedoptical density or radiolabeled signal when compared to control samplesfrom normal subjects correlates with a diagnosis of APS in a subject.

In an alternative exemplary embodiment of the competitive immunoassay,an indirect method using two antibodies is provided. β₂-GPI antigenspecific antibodies are added first as described in the precedingparagraph with the exception that they are not labeled. They areincubated with different concentrations of a sample from a subject. Aconstant amount of a second antibody is then added to the mixture of thesample and the first antibody. The second antibody recognizes constantregions of the heavy chains of the first antibody. For example, thesecond antibody may be an antibody which recognizes constant regions ofthe heavy chains of mouse immunoglobulin which has reacted with theβ₂-GPI antigens (anti-mouse immunoglobulin). The second antibody may belabeled with a fluorophore, chemilophore or radioisotope, as describedabove. Free labeled second antibody is separated from bound antibody. Ifan enzyme-labeled antibody is used, an appropriate substrate with whichthe enzyme label reacts is added and allowed to incubate. A decrease inoptical density or radioactivity from before and after addition of theserum sample in comparison with control samples is indicative thatautoantibodies in the serum sample have bound to the β₂-GPI antigens.Decreased optical density or radioactivity when compared to controlsamples from normal subject correlates with a diagnosis of a APS in asubject.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to autoimmune orchronic inflammatory disease markers is utilized.

In some embodiments, the β₂-GPI specific autoantibody level may be usedtogether with other biological markers as a panel for the diagnosis ofheart disease. The panel allows for the simultaneous analysis ofmultiple markers correlating with AAS. For example, a panel may includemarkers identified as correlating with AAS in a subject that is likelyor not to respond to a given treatment. Depending on the subject, panelsmay be analyzed alone or in combination in order to provide the bestpossible diagnosis and prognosis. Markers for inclusion on a panel areselected by screening for their predictive value using any suitablemethod, including but not limited to, those described in theillustrative examples below.

Data Analysis

In the present invention, a computer-based analysis program may also beused to translate the raw data generated by the detection assay intodata of predictive value for a clinician. The clinician can readilyaccess the predictive data using any suitable means. The clinician isthen able to immediately utilize the information in order to optimizethe care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information provides, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystem). Once received by the profiling service, the sample is processedand a profile is produced, specific for the diagnostic or prognosticinformation desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., likelihood of a liver disease suchas HCC to respond to a specific therapy) for the subject, along withrecommendations for particular treatment options. The data may bedisplayed to the clinician by any suitable method. For example, in someembodiments, the profiling service generates a report that can beprinted for the clinician (e.g., at the point of care) or displayed tothe clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or severity of disease.

EXAMPLES

Autoantibodies targeting β₂-Glycoprotein I (β₂-GPI), a component of theatherosclerotic plaque, are commonly found in patients with acuteischemic syndromes. Serum samples from APS (Antiphospholipid Syndrome)patients and from cardiovascular patients exhibiting acuteatherosclerotic syndromes were analyzed for IgG and IgA antibodies inboth anti-β₂-GPI and anti-cardiolipin (aCL) ELISA assays. All of the APSsamples used were positive in both assays. Serum samples from 382cardiovascular patients were also analyzed for IgG and IgA antibodies inthe same assays. In sharp contrast to the APS samples, it was found thatonly 1% of the samples from cardiovascular patients were positive forIgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive forIgA anti-β₂-GPI and only 1.6% for IgG anti-β₂-GPI. The antigenicspecificity of 29 serum samples from cardiovascular patients wasevaluated. Six different recombinant domain-deleted mutants (DM) ofhuman β₂-GPI and full-length human β₂-GPI (wild-type), were used incompetitive inhibition assays to inhibit the autoantibodies from bindingin the anti-β₂-GPI ELISA assays. Domain-deleted mutants D--345 andD---45 inhibited the binding in the IgA anti-β₂-GPI assay suggestingthat these autoantibodies recognize domain 4 of the β₂-GPI molecule.These results demonstrated that IgA anti-β₂-GPI autoantibodies fromatherosclerotic patients are distinct from IgA autoantibodies found inAPS samples.

Example 1

This example demonstrates that the predominant antibody profileexhibited by APS patients (cardiolipin IgG positive/β₂-GPI IgG/IgApositive) differed from the predominant profile exhibited byatherosclerotic patients with acute ischemic disease (cardiolipinIgG/IgA negative/β₂-GPI IgA positive) because of differing domainspecificity of the APS and atherosclerosis patient's antibodies. Using aseries of full-length β₂-GPI and β₂-GPI Dms, a large number of serumsamples from patients with APS and various atherosclerotic populationswere tested for IgG, IgA, and IgM antibodies to these constructs byusing a competitive inhibition ELISA. All specimens were also tested forIgG, IgA, and IgM aCL antibodies. This experiment demonstrated that 29of 29 IgA anti-β₂-GPI positive samples from atherosclerosis patientsspecifically recognized domain 4 of β₂-GPI.

Materials and Methods

Recombinant β₂-GPI

The recombinant β₂-GPI and β₂-GPI domain-deleted mutants (Dms) used areas previously described (Igarashi M, et al., Blood 87(8):3262-3270(1996). Briefly, TN5 insect cells were infected with high titer viralstock produced in Sf9 insect cells. Each construct contained a 6his tailthat was used for purifying the protein from culture media. Thenomenclature for domain deletion mutants uses numbers to indicate thepresence of domains while a dash symbolizes the domain is missing. ThusD--345 is the name given to the recombinant protein that containsdomains 3, 4 and 5 while lacking domains 1 and 2.

Patient Sample Selection

The diagnosis of each syndrome described was done according to clinicalpresentation, ultrasound, angiography or magnetic angioresonancestudies. Patients were enrolled consecutively in a tertiary center(University Hospital). Patients with infective endocarditis,osteonecrosis, neoplasms, cerebral hemorrhage, infection by HIV ortreponema pallidum, presence of known heritable causes of thrombosissuch as homocistinuria or factor V (Leiden) mutation, previous diagnosisof APS, or other connective tissue disorder (CTD) were excluded. Controlpatients were recruited from patients admitted to the Orthopedic clinicfor fractures or musculoligamentous disorders and without acutemyocardial infarction, stroke, or other cardiac conditions (Ranzolin A,et al., Arg Bras Cardiol, 83(2): 141-4; 137-140 (2004); Staub H L etal., Arg Bras Neuropsiquiat 61(3B): 757-63 (2003)).

A total of 511 archived specimens consisting of 382 sera fromindividuals with various atherosclerosis conditions and 129 sera fromindividuals with anti-phospholipid syndrome were studied. Theatherosclerosis group included sera from individuals with peripheralarterial disease (117), acute coronary syndrome (117), and acutemyocardial infarction (90). Ten samples were randomly selected from APSpatients that were positive for both IgG and IgA in the anti-β₂-GPIELISA, and 29 samples from atherosclerosis patients that were positivefor IgA in the anti-β₂-GPI ELISA.

Anti-β₂-GPI and Anti-Cardiolipin ELISA

All samples, both from the APS and cardiovascular patients, were testedfor the presence of anticardiolipin (aCL) antibodies and anti-β₂-GPIantibodies by ELISA. Specimens were first tested for the presence ofIgG, IgA, or IgM aCL and β₂-GPI antibodies using polyvalent aCL andanti-β₂-GPI screening ELISA tests. All ELISA kits used in this studywere manufactured by INOVA Diagnostics (INOVA Diagnostics, San Diego,Calif.) and run according to the manufacturer's instructions.

Competitive Inhibition ELISA

Tests were performed using the appropriate (IgG and/or IgA) anti-β₂-GPIELISA kit from INOVA Diagnostics. Each serum was titered to determinethe dilution required to give approximately 80% of maximum binding. Testinhibitors were diluted in the sample dilution buffer provided in thekits and 25 μl of each dilution or sample diluent alone was added to thewells. The serum samples were diluted in sample dilution buffer and 25μl of a constant dilution was added to the wells. The contents of thewells were mixed and plates were incubated at room temperature for 30minutes. Wells were washed 3 times with the wash buffer provided in thekits, 50 μl of the HRP conjugated anti-IgG or IgA added, incubated for30 minutes, washed 3 times with wash buffer and 50 μl of substratesolution was added. Wells were incubated at room temperature for 30minutes and 50 μl of stop solution was added. The OD 450 for each wellwas determined in an Anthos Labtec HT2 microplate reader (Salzburg,Austria). The percent inhibition was determined as follows: [(mean A₄₅₀obtained from the control wells without inhibitor less A₄₅₀ ofbackground)−(A₄₅₀ obtained in the presence of inhibitor less A₄₅₀ ofbackground)/mean A₄₅₀ obtained from the control wells without inhibitorless A₄₅₀ of background]×100.

Results

Anti-β₂-GPI and Anti-Cardiolipin

Serum samples from APS and cardiovascular patients were analyzed for IgGand IgA autoantibodies in both the anti-β₂-GPI and the anti-cardiolipin(aCL) assays. Almost 80% of the APS samples were positive by polyvalentIgG/IgA/IgM aCL and β₂-GPI screening assays (Table 1).

TABLE 1 Frequency of aCL and anti-β₂-GPI antibodies in APS andCardiovascular Groups by ELISA testing APS patients Cardiovascularpatients Total sera = 511 (n = 129) (n = 382) aCL Screen (IgG/IgA/IgM)78% 12% anti-β₂-GPI Screen 79% 46% (IgG/IgA/IgM) aCL IgG 64%  1% aCL IgA 9%  1% Anti-β₂-GPI IgG 43%  1% Anti-β₂-GPI IgA 48% 33%

Specific isotype testing of the APS sera revealed that approximately 64%were IgG and 9% were IgA ACA antibody positive, while 43% were IgG and48% were IgA anti-β₂-GPI positive. Serum samples from 370 cardiovascularpatients were similarly tested for total (IgG/IgA/IgM) and specific IgGand IgA antibodies in both the aCL and anti-β₂-GPI assays. In sharpcontrast to the APS samples, where IgG aCL and IgG anti-β₂-GPIantibodies were found in 64% and 43% of the specimens, respectively, itwas found that IgG aCL and IgG anti-β₂-GPI antibodies were present inonly 1% of the samples from cardiovascular patients. Even more strikingwas the observation that while the pattern of reactivity for IgA aCL andIgA anti-β₂-GPI was similar in the APS and cardiovascular patients (bothhad low levels of IgA aCL and moderate levels of IgA anti-β₂-GPI), IgAanti-β₂-GPI was the only major antibody present in the cardiovasculargroup. In contrast, the APL patients had moderate levels of antibodiesto IgG aCL, IgG anti-β₂-GPI, and IgA anti-β₂-GPI (Table 1).

Epitope(s) of β₂-GPI Recognized by Both IgG and IgA Anti-β₂-GPI I fromAPS Patients.

Recombinant β₂-GPI and two deletion mutants were used to determine theantigenic specificity of both the IgG and IgA autoantibodies from 10different APS patients. Each recombinant form of β₂-GPI was tested, in adose-dependent fashion, for its ability to inhibit these autoantibodiesfrom binding to full length β₂-GPI (Table 2, FIG. 1).

TABLE 2 Competitive inhibition assay using 10 different APS serumsamples with indicated recombinant B2GPI and deletion mutants. IgGAntibody D12345¹ D12--- D---45 Number Max² 50%³ Max 50% Max 50% 6612 899.5 98 24.0 17.0 >125 6626 88 31.8 90 88.0 3.5 >125 6635 95 11.8 96 29.53.0 >125 6647 92 25.8 94 66.0 5.0 >125 6656 64 38.4 67 85.0 10.0 >1256666 79 27.2 85 20.3 2.4 >125 6674 90 20.5 94 52.0 12.0 >125 7002 5853.7 53 178.0 7.0 >125 7005 77 23.5 71 58.0 1.9 >125 7010 83 15.8 8641.6 14.0 >125 IgA Antibody D12345 D12--- D---45 Number Max 50% Max 50%Max 50% 6612 80 13.1 74 36 28 >125 6626 62 42.4 39 >125 61 >125 6635 5744.7 67 29.6 0 >125 6647 68 30.7 58 66.4 0.7 >125 6656 74 24.3 42 >12540 >125 6666 48 67.3 42 >125 11 >125 6674 85 39.9 88 39.9 4 >125 7002 29116.3 21 >125 0 >125 7005 35 50.6 45 >125 40 >125 7010 63 16.7 59 5012 >125 >= Highest concentration tested. ¹= Domains included inconstruct. ²= Maximum Inhibition observed at concentrations tested. ³=Concentration (micromolar) to give 50% inhibition.

Only those constructs that contained domain 1 inhibited both the IgG andIgA autoantibodies. As shown in Table 2, both the IgG and IgAanti-β₂-GPI binding antibodies from all 10 patients were inhibited byboth constructs that contain domain 1. None of the samples wereeffectively inhibited, even at the highest concentration tested, by theconstruct that lacked domain 1. ID₅₀ values for mutants that containdomain 1 ranged from 1 to 50 μM for the IgG antibody and 13 to 100 μMfor the IgA antibody. By contrast the mutant that did not contain domain1 (D---45) did not effectively inhibit either the IgG nor the IgAantibody.

Epitope(s) of β₂-GPI Recognized by IgA Anti-β₂-GPI from Patients withAcute Cardiovascular Syndromes.

The differing β₂-GPI and aCL profile of the APS and cardiovascular sera(Table 1) suggested to us that the IgA anti-β₂-GPI antibodies incardiovascular patients may be distinct from those present in APSpatients and might target a different domain on the β₂-GPI protein.

Twenty nine samples from the cardiovascular patient cohort were selectedwhich were IgA anti-β₂-GPI antibody positive and aCL IgG, aCL IgM, andwith the exception of one sera, aCL IgA negative. The detailed β₂-GPIand aCL profiles of these sera are shown in Table 3.

TABLE 3 Anti-aCL and anti-β₂-GPI profile of samples from cardiovascularpatients¹ for inhibition study. β₂-GPI aCl aCl ACl β₂-GPI β₂-GPI IgM IgAIgG IgM Sample IgA Units IgG Units² Units Units Units Units ACS-52 58.90 10.1 n.t. n.t. n.t. ACS-53 20.7 0 4.0 n.t. n.t. n.t. ACS-54 33.1 9.6 0n.t. n.t. n.t. ACS-58 86.4 0 5.3 n.t. n.t. n.t. ACS-65 234.4 0 4.0 n.t.n.t. n.t. ACS-67 38.7 0 0 n.t. n.t. n.t. ACS-71 77.6 0 0 n.t. n.t. n.t.ACS-74 154.8 0 5.7 n.t. n.t. n.t. ACS-104 23.7 0 58.5 n.t. n.t. n.t.ACS-136 32.0 64.4 0.2 n.t. n.t. n.t. ACS-144 28.3 3.3 16.9 n.t. n.t.n.t. CAS-5 65.4 0 2.8 n.t. n.t. n.t. CAS-6 55.5 1.2 0 n.t. n.t. n.t.CAS-8 61.2 0 0 n.t. n.t. n.t. CAS-13 39.9 1.7 11.7 n.t. n.t. n.t. CAS-15130.1 0.1 0 n.t. n.t. n.t. CAS-18 35.5 0 0 n.t. n.t. n.t. CAS-28 60.5 00 n.t. n.t. n.t. CAS-29 51.8 4.1 5.7 n.t. n.t. n.t. MI-5 70.0 0 25.6n.t. n.t. n.t. MI-7 90.4 0 0 n.t. n.t. n.t. MI-10 183.9 0 25.4 n.t. n.t.n.t. MI-15 58.4 0 77.4 n.t. n.t. n.t. MI-37 32.7 0 41.2 n.t. n.t. n.t.MI-45 25.5 0 11.6 n.t. n.t. n.t. PAD-30 27.6 0 0 n.t. n.t. n.t. PAD-3922.1 0 0 n.t. n.t. n.t. PAD-42 45.5 0 0 27.9 n.t. n.t. PAD-101 26.4 3.025.4 n.t. n.t. n.t. n.t. = Specimens testing negative on aCL screeningassay were not tested on isotype-specific assays. ¹= ACS: Acute CoronarySyndrome. MI: Myocardial Infarction. CAS: Carotid Artery Study. PAD:Peripheral Artery Disease. ²= β₂-GPI results with negative values(resulting from extrapolation at bottom of the standard curve) wereassigned a value of 0.

Seven different recombinant β₂-GPI mutant proteins were used todetermine the antigenic specificity of the IgA β₂-GPI binding antibodiesfrom 29 different samples from patients with various cardiovascularconditions, including acute cardiac syndrome (11), acute myocardialinfarction (6), carotid artery disease (8), and peripheral arterydisease (4). Each mutant recombinant β₂-GPI protein was tested, in adose-dependent fashion, for its ability to inhibit the IgA antibody frombinding to full-length β₂-GPI (Table 4).

TABLE 4 Competitive inhibition assays using 29 different serum samples,from patients with cardiovascular problems¹, with indicated recombinantB2GP1 and deletion-mutants. 12345² 12--- 123-- 1234- ---345 ---45 ----5Ab# Max³ 50%⁴ Max 50% Max 50% Max 50% Max 50% Max 50% Max 50% ACS-104 6220 0 >125 13 >83 0 >63 79 30 76 24 10 >250 ACS-136 74 12 3 >125 0 >830 >63 72 6 76 26 3 >250 ACS-144 52 12 12 >125 12 >83 8 >63 63 6 68 79 >250 ACS-52 70 17 0 >125 14 >83 30 >63 86 6 87 2 21 >250 ACS-53 56 2315 >125 12 >83 24 >83 76 10 80 14 10 >250 ACS-54 71 5 11 >125 0 >830 >63 67 3 85 2 3 >250 ACS-58 57 24 0 >125 7 >83 24 >63 84 20 90 162 >250 ACS-65 82 12 0 >125 18 >83 88 5 92 4 94 8 0 >250 ACS-67 65 218 >125 2 >83 49 >63 75 21 83 23 20 >250 ACS-71 72 12 18 >125 7 >8313 >63 90 4 93 4 49 227 ACS-74 69 10 3 >125 2 >83 15 >63 90 5 91 129 >250 CAS-13 54 34 7 >125 0 >83 10 >63 74 17 72 33 15 >250 CAS-15 83 20 >125 1 >83 0 >63 92 2 91 17 11 >250 CAS-18 72 24 0 >125 13 >83 0 >6385 31 82 46 0 >250 CAS-28 65 18 7 >125 22 >83 39 >63 84 26 84 29 0 >250CAS-29 63 26 8 >125 17 >83 33 >63 76 36 75 28 0 >250 CAS-5 70 29 0 >12519 >83 29 >63 90 6 84 20 0 >250 CAS-6 68 24 14 >125 14 >83 27 >63 78 677 17 15 >250 CAS-8 75 23 14 >125 0 >83 6 >63 88 6 87 17 0 >250 MI-10 7414 8 >125 12 >83 64 43 90 13 89 4 0 >250 MI-15 71 16 6 >125 19 >8325 >63 71 34 80 12 19 >250 MI-37 37 52 0 >125 0 >83 0 >63 50 7 58 162 >250 MI-45 60 27 5 >125 16 >83 20 >63 67 38 66 8 0 >250 MI-5 73 1830 >125 0 >83 0 >63 90 2 58 5 0 >250 MI-7 85 2 0 >125 35 >83 48 55 76 2190 8 56 80 PAD- 27 >50 3 >125 0 >83 14 >63 67 51 72 55 6 >250 101 PAD-3057 24 0 >125 19 >83 58 41 78 30 79 24 0 >250 PAD-39 59 19 14 >125 10 >8354 46 75 30 77 24 0 >250 PAD-42 40 >50 9 >125 6 >83 9 >63 79 24 83 2914 >250 >= Greater than highest concentration tested ¹= ACS: AcuteCoronary Syndrome. MI: Myocardial Infarction. CAS: Carotid Artery Study.PAD: Peripheral Artery Disease. ²= Domains included in construct. ³=Maximum Inhibition observed at concentrations tested. ⁴= Concentration(μM) to give 50% inhibition.

An example of the results is shown graphically in FIG. 2. With theexception of the full-length construct, only the D--345 and D---45constructs inhibited these IgA antibodies. Four of the 29 samples werealso inhibited, albeit to a much lesser extent, by the D1234-construct.Only one of the samples was also inhibited by the D----5 construct. ID₅₀values for the D--345 and D---45 mutants ranged from 1 to 55 μM. Bycontrast, the D12--- and D123---mutants did not effectively inhibit thebinding of any of the 29 samples tested.

Discussion

It has been previously shown that the antigenic specificity of the IgGautoantibodies found in APS patients recognize domain 1 of the β₂-GPImolecule (Iverson, G M et al., PNAS 95: 15542-15546 (1998); Iverson, GM, et al., J. Immunol. 169:7097-7103 (2002)). The antigenic specificityof the IgA autoantibodies from APS patients however, was not known. Theinhibition studies reported here (FIG. 1, Table 2) clearly show that theantigenic specificity of the battery of 10 APS samples studied in thisreport are directed toward an epitope that is contained within domain 1of the β₂-GPI molecule. Thus, the antigenic specificity of both the IgGand IgA autoantibodies found in APS patients is domain 1.

This experiment also shows (FIG. 2 and Table 3) that IgA β₂-GPI bindingantibodies from patients with several types of AAS (acute myocardialinfarction, acute coronary syndrome, “carotid artery study”, andperipheral artery disease) recognize an epitope on domain 4 of theβ₂-GPI molecule. This should not be confused with earlier studies thatpurported to show autoantibodies from APS patients recognize domain 4 ofβ₂-GPI (Igarashi M., et al, Blood, 87 (8): 3262-70 (1996); George J, etal., J Immunol. 160(8):3917-3923 (1998)). These studies were designed tostudy the antigenic specificity of IgG, not IgA, autoantibodies.

The Dms that contained domain 4 inhibited in a similar, but notidentical pattern, among the various samples tested. For example, only 4were inhibited by the D1234-construct. This suggests that theseantibodies recognize comparable, but distinguishable, epitopes presenton domain 4. A recent molecular simulation derived from β₂-GPI crystalstructure supports this possibility. This study suggested 2discontinuous antigenic sequences in Domain 4β₂-GPI (Kasahara H, et al.,Int. Immunol. 17:1533-1542 (2005)). Domain 4 may have differentconformational states when present in constructs containing differentdomains. For example, a few samples recognized domain 4 when domain 5was absent, while the majority only recognized domain 4 when domain 5was present. Thus, these antibodies may recognize an epitope on domain 4that is affected by the presence of additional domains. Thisinterpretation was also supported by the simulation experiments.

Previously it has been shown that the orientation of β₂-GPI on the ELISAplate is important for the binding of anti-β₂-GPI autoantibodies whenmeasured by ELISA. (George J, et al., J. Immunol. 160(8):3917-3923(1998)) This could explain why these samples recognize β₂-GPI whenadsorbed onto plastic plates, but do not bind β₂-GPI when adsorbed ontocardiolipin. The binding of β₂-GPI to cardiolipin via domain 5 may givea different orientation than when bound to plastic. Binding via domain 5may alter either the availability of domain 4 or the configuration ofdomain 4, or both. However, the orientation of the β₂-GPI molecule whenadsorbed onto plastic is not completely understood. It is conceivablethat sufficient numbers of molecules adsorb to the plastic in such anorientation that domain 4 is neither hindered, that is available forantibody, nor has its conformation altered enough to negate the bindingof these antibodies.

Example 2

A clinical study was performed using an ELISA assay to detect domain4-specific antibodies using a domain 4/5 combined antigen using 218clinically characterized sera. The cohort included healthy controlindividuals (n=30), patients with cardiac stents (without symptoms,n=28; symptomatic, n=23), and patients with acute stroke (ischemicstroke and transient Ischemic attack; without intracerebral hemorrhage,n=137). The mean IgA antibody value for the healthy control populationwas 14.1 units, compared to 26.5 units for the patients with stents and29.5 for patients having an acute stroke (Table 5).

TABLE 5 ELISA assays using 218 different serum samples Number of seratested 30 51 137 Patient diagnosis Control Stent Acute stroke Mean valueof IgA Domain 4 14.09 26.46 29.54 and 5 of β₂-GPI antibodies

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the compositions, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes (for carrying outthe invention that are obvious to persons of skill in the art) areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference.

1. A method of detecting an autoantibody in a subject suspected ofhaving an acute atherosclerotic syndrome comprising the steps of: a)obtaining a serum or plasma sample from the subject, wherein the serumor plasma sample may contain IgA autoantibodies specific forβ₂-Glycoprotein I (β₂-GPI); b) contacting the serum or plasma samplewith a polypeptide consisting of β₂-GPI domain 4 and domain 5 to form acomplex between the polypeptide and the IgA autoantibodies; c) detectingthe IgA antibodies autoantibodies in the complex to establish a level ofIgA autoantibodies in the serum or plasma; and d) comparing the level ofIgA autoantibodies in the complex to a control level of IgAautoantibodies in serum or plasma from healthy control individuals;wherein if the level of IgA autoantibodies in the complex is greaterthan the control level, this indicates that the subject may have anacute atherosclerotic syndrome.